Our long-term goal is to understand molecular mechanisms underlying novel tumor suppressor genes Slit and USP33. Our previous work and preliminary data suggest that a prototypical neuronal repellent, Slit, is an important tumor suppressor for lung cancer and that Slit expression predicts better prognosis in lung cancer patients. To dissect Slit signaling pathways and examine mechanisms underlying tumor suppression in lung cancer, we examined how signal-transducing molecules interacted with Slit receptor Roundabout (Robo). We identified the deubiquitinating enzyme, USP33, as a critical component in Slit-Robo signaling pathway(s) in lung cancer. This proposal aims to examine Slit-Robo-USP33 mediated tumor suppression in lung cancer using molecular, biochemical and cell biological methods in combination with animal models and sequence analyses of human lung cancer samples. Our pilot deep-sequencing studies support the tumor suppressive function of Slit-Robo- USP33 pathway and the involvement of the downstream signaling molecules. We have established both in vitro assays and animal models to study the role of this newly uncovered tumor suppression pathway in lung cancer development. We plan to use these integrated molecular, cellular and genetic approaches to define the role of Slit-Robo signaling in suppressing lung cancer. Our proposed work will help elucidate molecular mechanisms underlying Slit and USP33 tumor suppressors and determine the role of genetic variations in Slit or USP33 and their downstream genes in development of human lung cancer.